In vitro studies on HIV (HIV-1) replication and neutralization are usually performed in human cell cultures supplemented with FBS instead of human serum (HS). Here we show that in contrast to FBS, addition of increasing amounts of human serum from noninfected donors to the cell culture directly correlates with an increase in HIV-1 replication in vitro. This effect is independent of cell line, virus strain, or batch of pooled human serum used. We found that human serum affects viral transcription in a dose-dependent manner by activating the activator protein-1 (AP-1) member proteins c-FOS, JunD, and JunB in TZM-bl cells. Analysis of the human serum component responsible for this effect indicates that it is a protein having a molecular mass between 250 and 300 kDa. This serum protein, HIV-1 enhancing serum protein (HESP), might promote viral transcription in vivo and consequently play a role in disease progression.